Methods using O6-alkylguanine-DNA alkyltransferases

Abstract

 Inter alia, a method for detecting a protein of interest in vivo, meaning in bacterial or eukaryotic cells, in cell culture or in cell extracts, in a cell in all compartments of a cell, on the surface of a cell or AGT fusion proteins pointing to the extracellular space, which comprises contacting an expressed fusion protein comprising the protein of interest and an O⁶- alkylguanine-DNA alkyltransferase (AGT) with a substrate having a label so that the AGT transfers the label from the substrate to become covalently bonded to the expressed fusion protein to permit detection thereof is disclosed.

Description

Field of the Invention

[0001] The present invention relates to methods of transferring a label from a substrate to a fusion protein comprising a protein of interest and an O⁶-alkylguanine-DNA alkyltransferase (AGT), and in particular to methods which further comprise detecting and/or manipulating the labelled fusion protein.

Background of the Invention

[0002] Progress in understanding complex biological systems depends on characterizing the underlying interactions of biomolecules, in particular proteins. While the DNA sequencing of an increasing number of organisms has identified their open reading frames (ORF), the possibilities to study the behaviour of the corresponding proteins in the living cell and to characterize multi-protein interactions in vivo and in vitro are limited. Most strategies that aim at realizing this objective are based on the construction of a fusion protein that, upon changes in the environment of the coupled protein, elicits a physical, physiological or chemical response. Examples include the yeast-two hybrid system, split-ubiquitin and green fluorescent protein (GFP) fusion proteins. However, all these techniques have various limitations or disadvantages.

[0003] German Patent Application No: 199 03 895 A (Kai Johnsson) describes an ELISA assay for the detection of O⁶-alkylguanine-DNA alkyltransferase (AGT). The mutagenic and carcinogenic effects of electrophiles such as N-methyl-N-nitrosourea are mainly due to the O⁶-alkylation of guanine in DNA. To protect themselves against DNA-alkylation, mammals and bacteria possess a protein, O⁶-alkylguanine-DNA alkyltransferases (AGT) which repairs these lesions [Pegg et al., 1995]. AGT transfers the alkyl group in a S_N2 reaction to one of its own cysteines, resulting in an irreversibly alkylated enzyme. As overexpression of AGT in tumour cells enables them to acquire drug resistance, particularly to alkylating drugs such as procarbazine, dacarbazine, temozolomide and bis-2-chloroethyl-N-nitrosourea, inhibitors of AGT have been proposed for use as sensitisers in chemotherapy [Pegg et al., 1995]. DE 199 03 895 A discloses an assay for measuring levels of AGT which relies on the reaction between biotinylated O⁶-alkylguanine-derivatives and AGT which leads to biotinylation of the AGT. This in turn allows the separation of the AGT on a streptavidin coated plate and its detection, e.g. in an ELISA assay. The assay is suggested for monitoring the level of AGT in tumour tissue, adjusting treatment using AGT inhibitors as sensitisers in chemotherapy and for use in screening for AGT inhibitors.

[0004] Damoiseaux, Keppler and Johnsson (ChemBiochem., 4: 285-287, 2001) discloses the modified O⁶-alkylated guanine derivatives incorporated into oligodeoxyribonucleotides for use as of chemical probes for labelling AGT, again to facilitate detecting the levels of this enzyme in cancer cells to aid in research and in chemotherapy. Two types of variant AGT substrates and an assay for AGT in which it is labelled with biotin (the same as that described in DE 199 03 895 A) are disclosed. In addition, the use of these O⁶-alkylated derivatives in the directed evolution of the AGT is suggested.

Summary of the Invention

[0005] Broadly, the present invention relates to a further use of O⁶-alkylguanine-DNA alkyltransferase (AGT) in a method of labelling, and optionally subsequently manipulating and/or detecting, a protein or peptide of interest in a system in which a fusion of the protein or peptide and AGT is contacted with a labelled substrate so that the AGT transfers the label from the substrate to the AGT fusion, thereby allowing the labelled AGT-protein fusion to be manipulated and or detected by virtue of the transferred label. This contrasts with the prior art uses of assays for measuring AGT levels in which the AGT is not present as a fusion protein.

[0006] Accordingly, in a first aspect, the present invention provides a method which comprises contacting a fusion protein comprising protein of interest and an O⁶-alkylguanine-DNA alkyltransferase (AGT) and a substrate having a particular label so that the AGT transfers the label so that it is covalently bonded to the fusion protein, wherein the label is an affinity tag, a first member of a specific binding pair which is capable of specifically binding to the second member of the specific binding pair, a molecule which is attachable to a solid phase, a molecule which is capable of generating reactive radicals, a candidate compound or library of candidate compounds, a molecule which is capable of crosslinking to other biomolecules, a nucleic acid or a derivative thereof capable of undergoing base-pairing with its complementary strand, a lipid, or a hydrophobic molecule with membrane-inserting properties. After transfer of the label to the fusion protein, the method may additionally involve the further step of detecting and/or manipulating the labelled fusion protein.

[0007] In one embodiment, the present invention provides a method of labelling a fusion protein comprising protein of interest and an O⁶-alkylguanine-DNA alkyltransferase (AGT), the method comprising contacting the fusion protein with a substrate having a particular label as defined hereinbefore so that the AGT transfers the label so that it is covalently bonded to the fusion protein.

[0008] In some embodiments, the method comprises one or more further steps, for example detecting and/or manipulating the labelled fusion protein.

[0009] In a further aspect, the present invention provides a method of detecting a fusion protein comprising protein of interest and an O⁶-alkylguanine-DNA alkyltransferase (AGT), the method comprising contacting the fusion protein with a substrate having a particular label as defined hereinbefore so that the AGT transfers the label so that it is covalently bonded to the fusion protein and detecting the protein construct using the label.

[0010] In a further aspect, the present invention provides a method of manipulating a fusion protein comprising protein of interest and an O⁶-alkylguanine-DNA alkyltransferase (AGT), the method comprising contacting the fusion protein with a substrate having a particular label as defined hereinbefore so that the AGT transfers the label so that it is covalently bonded to the fusion protein and manipulating the fusion protein using a physical and/or chemical property introduced by the label to the fusion protein.

[0011] In some embodiments of this aspect of the invention, the method may comprise detecting the protein construct using the label.

[0012] In a further aspect, the present invention provides a method of immobilising a fusion protein comprising protein of interest and an alkylguanine-DNA alkyltransferase (AGT) on a solid support, the method comprising contacting the fusion protein with a substrate having a label which is attachable to a solid support, wherein the AGT transfers the label so that it is covalently bonded to the fusion protein which thereby can be subsequently attached to the solid support. The method may involve the further step of contacting the labelled fusion protein with the solid support so that it becomes immobilised on the solid support. In this aspect of the invention, the label may be covalently attached to the solid support in a subsequent reaction, or may be one member of a specific binding pair, the other member of which is attached or attachable to the solid support, either covalently or by any other means (e.g. using the specific binding pair of biotin and avidin or streptavidin).

[0013] In a further aspect, the present invention provides a method to label AGT fusion proteins both in vivo as well as in vitro. The term in vivo labelling of a AGT fusion protein means labelling in all compartments of a cell as well as of AGT fusion proteins pointing to the extracellular space. If the labelling of the AGT fusion protein is done in vivo and the protein fused to the AGT is a plasma membrane protein, the AGT part of the fusion protein can be either attached to the cytoplasmic or the extracellular side of the plasma membrane. If the labelling is done in vitro, the labelling of the fusion protein can be either performed in cell extracts or with purified or enriched forms of the AGT fusion protein.

[0014] In a further aspect, the present invention provides a method of determining the interaction of a candidate compound or library of candidate compounds and a target substance or library of target substance. Examples of compounds and substances include ligands and proteins, drugs and targets of the drug, or small molecules and proteins. In this method, the protein of interest fused to the AGT comprises a DNA binding domain of a transcription factor or an activation domain of a transcription factor, a target substance or library of target substances is linked to the other of the DNA binding domain or the activation domain of the transcription factor, and the label is a candidate compound or library of candidate compounds suspected of interacting with the target substance(s).

[0015] In preferred embodiments, the method may further comprise transferring the candidate compound or library of candidate compounds to the AGT protein fusion and contacting the AGT fusion protein(s) labelled with the candidate compounds and the target substance(s) so that the interaction of a candidate compound joined to the AGT fusion protein and a target substance activates the transcription factor. The activated transcription factor can then drive the expression of a reporter which, if the method is carried out in cells, can be detected if the expression of the reporter confers a selective advantage on the cells. In some embodiments, the method may involve one or more further steps such as detecting, isolating, identifying or characterising the candidate compound(s) or target substance(s).

[0016] In the present application, the O⁶-alkylguanine-DNA alkyltransferase or 'AGT' has the property of transferring a label present on a substrate to one of the cysteine residues of the AGT forming part of a fusion protein. In preferred embodiments, the AGT is an O⁶-alkylguanine-DNA alkyltransferase, for example human O⁶-alkylguanine-DNA alkyltransferase which is described in Pegg et al. 1995 and references therein. However, other alkylguanine-DNA alkyltransferases are known, e.g. murine or rat forms of the enzyme described in Roy et al., 1995, which can be employed in the present invention provided that they have the property defined above. In the present invention, O⁶-alkylguanine-DNA alkyltransferase also includes variants of a wild-type AGT which may differ by virtue of one or more amino acid substitutions, deletions or additions, but which still retain the property of transferring a label present on a substrate to the AGT or the protein or peptide with which it forms a fusion. Other variants of AGTs may be chemically modified using techniques well known to those skilled in the art. AGT variants may be produced using protein engineering techniques known to the skilled person and/or using molecular evolution to generate and select new O⁶-alkylguanine-DNA alkyltransferases.

[0017] In the present invention, the reference to the protein part of the fusion protein with the AGT is intended to include proteins, polypeptides and peptides of any length and both with and without secondary, tertiary or quaternary structure. Examples of applications of the present invention are provided below.

[0018] In the present invention, the labelled substrate is preferably a labelled benzylguanine substrate, and more preferably is an O⁶-benzylguanine derivative. An example of such a derivative is an O⁶-benzylguanine derivative that is derivatised at the 4-position of the benzyl ring with the following general formula:

wherein:

R¹ is a proton, a β-D-2'-deoxyribosyl, or a β-D-2'-deoxyribosyl that is part of an oligodeoxyribonucleotide, preferably having a length between 2 and 99 nucleotides;

R² is a linker group, for example a flexible linker such as a substituted or unsubstituted alkyl chain, a polyethylene glycol; and

label is a molecule as defined hereinbefore responsible for the detection and/or manipulation of the fusion protein.

[0019] Examples of modified O⁶-benzylguanine derivatives suitable for use in accordance with the present invention are provided in Figure 1. Further, the present inventors have found that the AGT can tolerate a considerable degree of flexibility in the identity of the substrate, allowing a wide range of substrates to be used with the following general formula:

wherein:

R¹ is a group accepted by AGT, allowing the AGT to transfer the label to the AGT-protein fusion, for example a substituted or unsubstituted alkyl chain, a substituted or unsubstituted cycloalkyl group with a ring size between three and ten carbons, a substituted or unsubstituted heterocycle with a ring size between three and ten carbons, a substituted or unsubstituted aromatic heterocycle with a ring size between three and ten carbons;

R² is a linker group, for example a flexible linker of varying length such as a substituted or unsubstituted alkyl chain or a polyethylene glycol; and

R³ is a proton, a β-D-2'-deoxyribosyl, or a β-D-2'-deoxyribosyl that is part of an oligodeoxyribonucleotide, preferably having a length between 2 and 99 nucleotides; and

label is a molecule as defined hereinbefore responsible for the detection and/or manipulation of the fusion protein.

[0020] The label part of the substrate can be chosen by those skilled in the art dependent on the application for which the fusion protein is intended. Examples of labels include:

(1) A molecule which is one part of a specific binding pair which is capable of specifically binding to a partner. Such specific binding pairs are well known in the art and include, for example, biotin, which can bind to avidin or streptavidin;

(2) A molecule that is suspected to interact with other biomolecules;

(3) A library of molecules that are suspected to interact with other biomolecules;

(4) A molecule which is capable of crosslinking to other biomolecules as known to those skilled in the art [Nadeau et al., 2002];

(5) A molecule which is capable of generating hydroxyl radicals upon exposure to H₂O₂ and ascorbate such as a tethered metal-chelate [Hori et al., 2002];

(6) A molecule which is capable of generating reactive radicals upon irradiation with light such as malachite green [Jay et al., 1999];

(7) A nucleic acid or a derivative thereof capable of undergoing base-pairing with its complementary strand;

(8) A lipid or other hydrophobic molecule with membrane-inserting properties;

(9) A biomolecule with desirable enzymatic, chemical or physical properties;

(10) A molecule possessing a combination of any of the properties listed above.

[0021] By way of example, embodiments of the present invention will now be described in more detail with reference to the accompanying figures.

Brief Description of the Figures

[0022] 

Figure 1: A) Mechanism of O⁶-alkylguanine-DNA alkyltransferase; B) Structure of O⁶-benzylguanine; C) General structure of O⁶-benzylguanine derivatives used in the examples; D) General scheme for labeling of AGT fusion proteins, X being the protein fused to AGT; E) Structures of AGT substrates used in the examples. The sequence of the oligonucleotide (22mer) is: 5'-GTGGTGGGCGCTGXAGGCGTGG-3' where X = BG-Bt (SEQ ID NO:1).

Figure 2: Western blots after labelling of AGT fusion proteins in vivo. A) Western-Blot of total cell extract of E. coli expressing Pep-hAGT with and without BG-Bt in the medium.

A streptavidin-peroxidase conjugate is used to detect biotinylated proteins. The band at 20 kD corresponds to a protein that is biotinylated in E. coli in the absence of BG-Bt. B) Western-Blot of total cell extract of yeast expressing a hAGT-DHFR-HA fusion protein with and without BG-DIG in the medium. An anti digoxigenin-peroxidase conjugate is used to detect digoxigenin-labelled proteins.

Detailed Description

[0023] The following applications of the present invention are provided by way of examples and not limitation. The method disclosed herein is generally applicable to a range of applications and is capable of specifically and covalently labelling fusion proteins with (1) labels which are capable of sensing and inducing changes in the environment of the labelled fusion protein and/or (2) labels which aid in manipulating the fusion protein by the physical and/or chemical properties specifically introduced by the label to the fusion protein. The method disclosed herein can be used to label AGT fusion proteins both in vivo and in vitro.

[0024] The present invention is based on the realisation that specific attachment of a label to a desired protein could be carried out by constructing a fusion protein between that protein of interest and taking advantage of the mechanism of an O⁶-alkylguanine-DNA alkyltransferase such as human DNA repair enzyme O⁶-alkylguanine-DNA alkyltransferase (hAGT). This enzyme irreversibly transfers the alkyl group from its substrate, O⁶-alkylguanine-DNA, to one of its cysteine residues (Figure 1). A substrate analogue that rapidly reacts with hAGT is O⁶-benzylguanine, the second order rate constant being approximately 10³ sec^-1 M^-1 (Figure 1). We have shown that substitutions of O⁶-benzylguanine at the C4 of the benzyl ring do not significantly affect the reactivity of hAGT against O⁶-benzylguanine derivatives. This enables the use of O⁶-benzylguanine derivatives that have a label attached to the C4 of the benzyl ring to covalently and specifically attach the label AGT fusion proteins in vivo or in vitro (Figure 1D). The labelling is independent of the nature of the fusion protein.

[0025] If the labelling is done in vivo in cell culture or in cell extracts, the labelling of the endogenous AGT of the host is advantageously taken into account. If the endogenous AGT of the host does not accept O⁶-benzylguanine derivatives or related compounds as a substrate, the labelling of the fusion protein is specific. In mammalian cells (human, murine, rat), labelling of endogenous AGT is possible. In those experiments where the simultaneous labelling of the endogenous AGT as well as of the AGT fusion problem poses a problem, previously described AGT-deficient cell lines can be used [Kaina et al., 1991]. In general, the present invention can be employed in all applications of the technique were the covalent and specific attachment of a label to a AGT fusion protein is used to monitor or influence the behaviour of the AGT fusion protein or is used to manipulate the AGT fusion protein by virtue of the introduced label. Examples of applications for the use of this technology follow.

[0026] The use of a labelled AGT substrates, such as O⁶-benzylguanine derivatives, where the substrate carries a detectable label which can be transferred to the AGT allows the present invention to be used to specifically and covalently attach the detectable label to the AGT fusion protein, either in a cell, on the surface of a cell (in vivo) or in vitro. This allows the detection and characterization of the AGT fusion protein in vivo or in vitro. The term in vivo includes labelling in all compartments of a cell as well as of AGT fusion proteins pointing to the extracellular space.

1) The label as a tag to detect and isolate AGT fusion proteins

[0027] The use of AGT substrates, such as O⁶-benzylguanine derivatives, which are labelled with an affinity tag such as biotin allows the present invention to be used to transfer an affinity tag to the AGT-protein fusion, thereby allowing the fusion protein to be bound by a binding partner of the affinity tag. By way of example, the addition of AGT substrates labelled with an affinity tag such as biotin to cells (bacterial or eukaryotic) expressing an AGT fusion protein, or to the cell extracts of such cells or to purified AGT fusion proteins, will lead to the covalent modification of the fusion protein with the affinity tag. This will then allow the isolation of the fusion protein using the interaction between the affinity tag and its binding partner, e.g. in the case of biotin, immobilized avidin or streptavidin. If the label is linked to the AGT-protein fusion via a linker containing a cleavable bond, such as a disulfide bridge, or if the linker is photocleavable, the AGT fusion protein can be released from the affinity tag after its isolation.

2) The label as of source of reactive radicals

[0028] AGT substrates, such as O⁶-benzylguanine derivatives, can be used to introduce labels into AGT-protein fusions which are capable of generating reactive radicals, such as hydroxyl radicals, upon exposure to an external stimuli. The generated radicals can then inactivate the AGT fusion proteins as well as those proteins that are in close proximity of the AGT fusion protein, allowing the study the role of these proteins. Examples of such labels are tethered metal-chelate complexes that produce hydroxyl radicals upon exposure to H₂O₂ and ascorbate, and chromophores such as malachite green that produce hydroxyl radicals upon laser irradiation. The use of chromophores and lasers to generate hydroxyl radicals is also known in the art as chromophore assisted laser induced inactivation (CALI) [Jay et al., 1998]. CALI is a method that is used to specifically inactivate certain proteins within a cell in a time-controlled and spatially-resolved manner and which is based upon the spatial neighbourhood of a chromophore and a protein.
Upon laser irradiation the chromophore generates hydroxyl radicals, which inactivate all proteins within and only within about 0.1 nm of the chromophore. So far, the chromophore is brought in the spatial neighbourhood of the protein of interest by microinjecting chromophore-labelled antibodies specific to the protein of interest. In the present invention, labelling AGT fusion proteins with chromophores such as malachite green and subsequent laser irradiation would allow to inactivate the AGT fusion protein as well as those proteins that interact with the AGT fusion protein in a time-controlled and spatially-resolved manner. The method can be applied both in vivo or in vitro.

[0029] In a similar manner, AGT fusion proteins can be labelled with tethered metal-chelates and the AGT fusion protein and those proteins that interact with the AGT fusion protein can be inactivated in a specific manner upon exposure to H₂O₂ and ascorbate. The method can not only be used to study the function of an AGT fusion protein or those that are in close proximity of the AGT fusion protein, but also to identify those proteins that are in close proximity of a AGT fusion protein. Here, proteins which are in close proximity of the AGT fusion protein can be identified as such by either detecting fragments of that protein by a specific antibody, by the disappearance of those proteins on a high-resolution 2D-electrophoresis gels or by identification of the cleaved protein fragments via separation and sequencing techniques such as mass spectrometry or protein sequencing by N-terminal degradation.

3) The label as a ligand mediating protein-protein interactions

[0030] The use of labelled AGT substrates, such as O⁶-benzylguanine derivatives can be used to transfer a ligand to the AGT-protein fusion. This allows binding partners of the ligand, such as proteins, to bind to the AGT-protein fusion. For example, where the label is a ligand which is capable of binding to a binding partner, contacting such a substrate with a AGT fusion protein will lead to specific attachment of the ligand to the fusion protein. If the ligand binds to another protein Y and the dimerization of the protein Y with the labelled AGT fusion protein leads to a biological function or a measurable signal, the biological function or the measured signal depends on the addition of the AGT substrate carrying the label. A specific example would be the use of AGT substrates and AGT fusion protein in the so-called three-hybrid system described by Ho et al., 1996, to regulate gene expression with small molecules. In this case, AGT is fused to the DNA-binding domain of a transcription factor. A protein Y, such as FKBP that binds a ligand, such as FK506, is fused to the activation domain of a transcription factor. Supplying the cells with the AGT substrate that carries the ligand, in this example FK506, will lead to the formation of a functional transcription factor and gene expression.

4) The label as a drug or biological active molecule whose target is unknown

[0031] The use of O⁶-benzylguanine derivatives or related AGT substrates that carry a label and where the label is a drug or a biological active small molecule that binds to an yet unidentified protein Y. Here the goal would be to identify the target protein Y of the biological active molecule. In this case, AGT is fused to the DNA-binding domain of a transcription factor. A cDNA library of the organism which expresses the unknown target protein Y is fused to the activation domain of a transcription factor. Adding the O⁶-benzylguanine derivatives or related AGT substrate that carry a label and where the label is the drug or the biological active small molecule will lead to the formation of a functional transcription factor and gene expression only in the case where this molecule binds to its target protein Y present in the cDNA library and fused to the activation domain. If gene expression is coupled to a selective advantage, the corresponding host carrying the plasmid with the target gene Y of the drug or bioactive molecule can be identified.

5) The label as a library of small molecules to identify molecules that bind to the protein Y

[0032] The use of O⁶-benzylguanine or related AGT substrates that carry a label and where the label is a library of chemical molecules: Here the goal would be to identify small molecules that bind to a protein Y under in vivo conditions, which might be a potential drug target. In this case, AGT is fused to the DNA-binding domain of a transcription factor. The target protein Y is fused to the activation domain of a transcription factor. Adding a library of small molecules attached as label to a O⁶-benzylguanine derivative will lead to the formation of a functional transcription factor and gene expression only in the case where the label (i.e. the small molecule) binds to its target protein Y fused to the activation domain. If gene expression is coupled to a selective advantage, those molecules of the library leading to the growth of the host can be identified.

6) The use of BG derivatives to immobilize AGT fusion proteins and/or to create protein arrays of AGT fusion proteins

[0033] The use of O⁶-benzylguanine derivatives or related AGT substrates carrying a label and where the label is a molecule that can be bound non-covalently by another molecule that is itself attached to the surface. An example is where the label is biotin and the molecule attached to the surface is streptavidin or avidin. Possible examples for a carrier would be either a glass side, a microtiter plate or any functionalized polymer. The immobilization of the AGT substrate via its label allows the subsequent immobilization of a AGT fusion protein on the carrier by the transfer of the label to the fusion protein. Spotting (different) AGT fusion proteins in a spatially resolved manner on the carrier allows the creation of protein arrays.

7) The label as a cross-linker to detect proteins that interact with the AGT fusion protein

[0034] The use of O⁶-benzylguanine derivatives or related AGT substrates which carry a label and where the label is a molecule that can cross-link to other proteins. Examples of such cross-linkers are molecules containing functional groups such as maleimides, active esters or azides and others known to those proficient in the art and described in Nadeau et al., 2002. Contacting such AGT substrates with AGT fusion proteins that interact with other proteins (in vivo or in vitro) can lead to the covalent cross-linking of the AGT fusion protein with its interacting protein via the label. This allows the identification of the protein interacting with the AGT fusion protein.

Examples

[0035] The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to practice the invention, and are not intended to limit the scope of the invention.

Example A: Covalent labelling of AGT fusion proteins in E. coli

[0036] The following example, the labelling of Pep-hAGT in E. Coli using BG-Bt, demonstrates the feasibility of covalently labelling hAGT fusion proteins in E. coli. The sequence of the peptide fused to the N-terminus of hAGT (yielding Pep-hAGT) is (in single letter code) MHHHHHHSSA (SEQ ID NO:2) followed by the first amino acid of hAGT, a methionine. Liquid cultures of XL-1 Blue E. coli cells containing a pET-15b (Novagen) based expression vector encoding hAGT with an N-terminal fusion peptide, i.e. Pep-hAGT, were grown to an optical density OD_600nm of 0.6. Expression of Pep-hAGT was induced by adding IPTG to a final concentration of 1 mM. At the same time BG-Bt was added to a final concentration of 10 µM and the bacteria were incubated for 2 h at 37°C. Cells were harvested by centrifugation and the pellet washed twice to remove access BG-Bt. A resuspended aliquot of cells was analysed by Western Blotting. Biotinylated proteins were detected using a streptavidin-peroxidase conjugate (NEN) and a chemiluminescent peroxidase substrate (Renaissance reagent plus, NEN) (Figure 2).

Example B: Covalent labelling of AGT fusion proteins in yeast

[0037] The following example demonstrates the feasibility of covalently labelling hAGT fusion proteins in yeast. Here, a hAGT-DHFR-HA fusion protein is biotinylation in yeast using BG-Bt. The fusion protein is constructed on the DNA level using standard molecular biology procedures. In short, the stop codon of hAGT is replaced by codons for the amino acids RSGI, which are then followed by the codon for the first amino acid of DHFR from mouse, a Met [Nunberg et al., 1980]. The codons for the linker between hAGT and DHFR also encode for a Bgl II site, its DNA sequence being AGATCT. To construct the fusion between DHFR and the HA tag, the stop codon of DHFR is replaced by a codon for the first amino acid of the HA-tag [Kolodziej, 1991]. The HA-tag is followed by a stop codon. A culture of L40 yeast cells, containing the expression vector p314AK1 in which the hAGT-DHFR-HA protein is under control of the p_cup1 promoter, was grown to an OD₆₀₀ of 0.6. Expression of hAGT-DHFR-HA was induced by adding CuSO₄ to a concentration of 100 µM and BG-Bt was simultaneously added to a concentration of 10 µM. Aliquots were taken after 2.5 h and 5 h and cells harvested by centrifugation. The pellet was washed twice to remove remaining BG-Bt. After lysis of the yeast cells by freeze/thaw cycling the cell extract was analysed for the presence of biotinylated hAGT-DHFR-HA fusion protein using an ELISA. In short, the biotinylated hAGT-DHFR-HA was immobilized in streptavidin-coated microtiter wells and detected by using an antiHA-antibody (Babco) as a primary and an antimouse-HRP conjugate (Sigma) as a secondary antibody (Figure 1) [Kolodziej, 1991]. The ELISA was developed using the peroxidase substrate ABTS and the signal (absorbance) measured at OD_405nm. The signal for the in vivo biotinylated hAGT-DHFR-HA fusion protein was at least fivefold above background. The background signal was defined as the OD_405nm of cell lysates obtained from cells treated exactly as above but omitting the addition of BG-Bt.

Example C: Covalent labelling of hAGT fusion proteins in yeast

[0038] The following example demonstrates the feasibility of covalently labelling hAGT fusion proteins in yeast. Here, the hAGT-DHFR-HA fusion protein is labelled with digoxigenin in yeast using BG-DIG. The construction of the hAGT-DHFR-HA fusion is described in example B. A culture of L40 yeast cells containing the expression vector p314AK1 in which the gene of a hAGT-DHFR fusion protein is under control of the p_cup1 promoter was grown to an OD_600nm of 1.2. Expression of the hAGT-DHFR fusion protein was induced by adding CuSO₄ to a concentration of 100 µM and BG-DIG was simultaneously added to a concentration of 20 µM. After 2 h cells from 1 ml of shake-flask culture were harvested by centrifugation. The pellet was washed three times with medium to remove remaining BG-DIG. After lysis of the yeast cells by freeze/thaw cycling the cell extract was analysed for the presence of digoxigenated hAGT-DHFR fusion protein by Western blotting. Digoxigenated proteins were detected using an anti-digoxigenin-peroxidase conjugate (Roche) and a chemiluminescent peroxidase substrate (Renaissance reagent plus, NEN) (Figure 2).

Example D: Covalent labelling of hAGT fusion proteins in human cell lines

[0039] The following example demonstrates the feasibility of labelling AGT fusion proteins in mammalian cells. Here, endogenous hAGT in human cells (HEK 293) is labelled with fluoresceine using BG-AcFc. Although this example describes the use of a spectroscopic probe, it can be likewise performed with a particular label of the invention. HEK 293 cells were incubated with 5 µM BG-AcFc in PBS for 5min. The acetylated fluoresceine derivative BG-AcFc is cell-permeable and non-fluorescent but can be expected to be hydrolysed rapidly within the cell to yield the fluorescent BG-Fc. The cells were then washed by changing the PBS to remove any access substrate BG-AcFc and incubated in PBS for 20 min. Images were then taken with a confocal fluorescence microscope (Ext. 492 nm; Em. 510 nm). As a control experiment, the HEK 293 cells were treated as above but incubated prior to addition of BG-AcFc overnight with O⁶-benzylguanine (1 µM). This should inactivate the endogenous hAGT and therefore prevent the accumulation of the fluorescence in the nucleus. As expected, no accumulation of fluorescence in the nucleus was observed when the cells were preincubated with O⁶-benzylguanine. To independently confirm that the hAGT accepts BG-Fc as a substrate, recombinant Pep-hAGT (10 µM, as described in example A was incubated with 100 µM BG-Fc at 25°C in 50 mM Tris-Cl, 10 mM DTT, 1 mM EDTA, 200 µg/ml BSA, 10% Glycerol, pH 7.4 for 10 minutes, followed by addition of 900 µl PBS (phosphate buffered saline: 137 mM NaCl, 2.7 mM KCI, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4). Separation of excess substrate BG-Fc was achieved by gel filtration on a NAP^™-10 Column (Pharmacia) according to the supplier's instruction. The Pep-hAGT was then characterized in a standard fluorescence spectrophotometer. The sample was excitated at 222, 238 and 490 nm, respectively and showed maximal emission at a wavelength of 523 nm, verifying that the protein was labelled with fluoresceine. A solution of 20 nM BG-Fc in PBS was measured as reference. The substrate's emission wavelength is 519 nm (excitation at 237, 323 and 490 nm respectively).

Example E: Covalent labelling of AGT fusion proteins in cell extracts

[0040] To demonstrate that fusion proteins with AGT can be directly labelled and manipulated (here immobilized) in cell extracts the following N- and C-terminal fusion proteins with hAGT proteins were constructed via standard molecular cloning procedures and cloned into a yeast expression vector:

(i) V5-NLS-B42-hAGT, where V5 stands for V5 epitope, NLS for SV40 large T antigen nuclear localization sequence and B42 stands for an artificial transcriptional activator B42 [Ma et al., 1987]. The last codon of the B42 transactivation domain is followed by the 22 amino acid sequence ASKKGTELGSTTSNGRQCAGIL (SEQ ID NO:3). The last three codons include a Eco RI site for the C-terminal cloning of the hAGT to B42. A Not I site is the C-terminal restriction site for the hAGT, whose sequence includes a stop codon;

(ii) hAGT-HA-Ura3, where Ura3 stands for the yeast enzyme orotic acid decarboxylase and HA stands for the Ha epitope. Here, the stop codon for the hAGT is replaced by RS linker followed by the first amino acid of the HA-tag. The HA-tag is directly followed by the Ura3 gene;

(iii) hAGT-DHFR-HA, where DHFR stands for the mouse dihydrofolate reductase and HA stands for the Ha epitope. Construction see example B; and

(iv) SSN6-hAGT, where SSN6 stands for a yeast repressor of DNA transcription [Schultz et al., 1987]. Here, the stop codon of hAGT is replaced by codons for the amino acids RSGSG, which are then followed by the codon for the first amino acid of SSN6 of yeast, a methionine.

[0041] The expression of all genes were controlled by the p_CUP1 promoter. L40 yeast cells containing an expression vector encoding for one of the fusion protein were grown to an OD of 0.6 and expression of the fusion protein was induced by adding CuSO₄ to a concentration of 100 µM. Aliquots (2 ml) were taken after 5 h and cells harvested by centrifugation. After lysis of the cells by freeze/thaw cycling the yeast extract was incubated with BG-Bt-oligo (10 pmol) for 20 min at room temperature, leading to biotinylation of the fusion protein. Subsequently, the suspension was transferred into a streptavidin-coated microtiter plates (Roche molecular biochemicals) and incubated for 1 h. After extensive washing of the well with PBS, the immobilized fusion protein was detected using either an anti-HA-antibody (Babco) or an anti-hAGT-antibody (in the case of the SSN6-hAGT fusion protein) as a primary and an antimouse-peroxidase conjugate (Sigma, #A4416) as a secondary antibody and subsequent incubation with the peroxidase substrate ABTS using standard biochemical procedures. In all cases, the signal which was measured as the OD_405nm was at least five-fold above background. Background was measured for each fusion protein by omitting the addition of BG-Bt-oligo to the cell extracts.

References

[0042] The references are in alphabetic order.

RB Ali et al., Molecular and Cellular Biology, 18, 1660-1669 (1998)

R Damoiseaux, A Keppler and K Johnsson, ChemBiochem, 4, 285-287 (2001)

SN Ho et al., Nature 382, 822-6 (1996)

R Hori and N Baichoo in Protein-Protein interactions: a molecular cloning manual; Ed. E Golemis, Cold Spring Harbor Laboratory Press; pp. 288-311 (2002)

DG Jay and T Sakurai, Biochim. Biophys. Acta M39-48 (1999)

Kaina et al., Carcinogenesis 12, 1857-67 (1991)

PA Kolodziej and RA Young, Methods Enzymol. 194, 508-19 (1991)

J Ma and M Ptashne, Cell 51, 113-9 (1987)

OW Nadeau and GM Carlson in Protein-Protein interactions: a molecular cloning manual; Ed. E Golemis, Cold Spring Harbor Laboratory Press; pp. 75-92 (2002)

JH Nunberg et al., Cell 19, 355-364 (1980)

AE Pegg et al., Prog Nucleic Acid Res Mol Biol. 51, 167-223 (1995)

J Schultz, M Carlson Mol Cell Biol 7, 3637-45 (1987)

[0043] Certain aspects of the present inventive concept may be indicated as follows:

1. A method performed in vitro or in cell culture which comprises contacting a fusion protein comprising protein of interest and an O⁶-alkylguanine-DNA alkyltransferase (AGT) with a substrate having a label so that the AGT transfers the label so that it becomes covalently bonded to the fusion protein, wherein the label is an affinity tag, a first member of a specific binding pair which is capable of specifically binding to the second member of the specific binding pair, a molecule which is attachable to a solid phase, a molecule which is capable of generating reactive radicals, a candidate compound or library of candidate compounds, a molecule which is capable of crosslinking to other biomolecules, a nucleic acid or a derivative thereof capable of undergoing base-pairing with its complementary strand, a lipid, or a hydrophobic molecule with membrane-inserting properties.

2. The method according to 1, wherein the label is an affinity tag, a first member of a specific binding pair which is capable of specifically binding to the second member of the specific binding pair, or a molecule which is attachable to a solid phase.

3. The method according to 1 or 2, which comprises detecting the labelled fusion protein in an in vitro system.

4. The method according to 1 or 2, which comprises detecting the labelled fusion protein in a cell culture system.

5. The method of 4, further comprising the initial step of transforming the cells with an expression vector comprising nucleic acid encoding the fusion protein linked to control sequences to direct its expression.

6. The method according to any one of the preceding , further comprising manipulating the labelled fusion protein using a property introduced by the label to the fusion protein.

7. The method according to anyone of the preceding , wherein the labelled substrate is incorporated into a nucleic acid molecule, and the nucleic acid molecule is an oligonucleotide between 2 and 99 nucleotides in length.

8. The method according to any one of the preceding , wherein the labelled substrate is a benzyl guanine substrate.

9. The method according to 8, wherein the labelled benzyl guanine substrate is represented by the general formula:

wherein:

R¹ is a proton, a β-D-2'-deoxyribosyl or a β-D-2'-deoxyribosyl that is part of an oligodeoxyribonucleotide;

R² is a linker group; and

label represents a group for use in detecting and/or manipulating the fusion protein.

10. The method according to 8, wherein the labelled benzyl guanine substrate is represented by the general formula:

wherein:

R¹ is a group accepted by AGT allowing the AGT to transfer the label to the protein fusion;

R² is a linker group;

R³ is a proton, a β-D-2'-deoxyribosyl, or a β-D-2'-deoxyribosyl that is part of an oligodeoxyribonucleotide; and
label represents a group for use in detecting and/or manipulating the fusion protein.

11. The method according to 10, wherein R¹ is a substituted or unsubstituted alkyl chain, a substituted or unsubstituted cycloalkyl group with a ring size between three and ten carbons, a substituted or unsubstituted heterocycle with a ring size between three and ten carbons, or a substituted or unsubstituted aromatic heterocycle with a ring size between three arid ten carbons.

12. The method according to any one of 9 to 11, wherein the linker group R² is a substituted or unsubstituted alkyl chain or a polyethylene glycol.

13. The method according to any one of 9 to 12, wherein the oligodeoxyribonucleotide has a length between 2 and 99 nucleotides.

14. The method according to any one of 1 to 13, wherein the label is an affinity tag.

15. The method according to any one of 1 to 13, wherein the label is a first member of a specific binding pair which is capable of specifically binding to the second member of the specific binding pair.

16. The method according to any one of 1 to 15, wherein the label is a molecule which is attachable to a solid phase.

17. The method according to 14, 15 or 16, wherein the label is biotin, avidin or streptavidin.

18. The method according to 14, 15 or 16, wherein the label is linked to the fusion protein via a cleavable linker so that the fusion protein can be released from the label.

19. The method according to 14, 15 or 16, wherein the label is linked to the fusion protein via a photocleavable linker so that the fusion protein can be released from the label.

20. The method according to 15, further comprising contacting the fusion protein with a molecule comprising the second member of the specific binding pair.

21. The method according to 15 or 20, wherein the first and second members of the specific binding pair are a first and second protein, an antibody and antigen, an enzyme and substrate or a ligand and receptor.

22. The method according to 16, wherein the method comprises the further step of contacting the labelled fusion protein with the solid support so that it becomes immobilised on the solid support.

Claims

1. A method for detecting a protein of interest in vivo, meaning in bacterial or eukaryotic cells, in cell culture or in cell extracts, in a cell in all compartments of a cell, on the surface of a cell or AGT fusion proteins pointing to the extracellular space, which comprises contacting an expressed fusion protein comprising the protein of interest and an O⁶- alkylguanine-DNA alkyltransferase (AGT) with a substrate having a label so that the AGT transfers the label from the substrate to become covalently bonded to the expressed fusion protein to permit detection thereof.

2. A method according to claim 1 wherein the substrate is a labelled benzyl guanine substrate represented by the general formula:

wherein

R¹ is a proton;

R² is a linker group;
and
label represents a group for use in detecting and/or manipulating the fusion protein;
or by the general formula:

wherein

R¹ is a group accepted by AGT allowing the AGT to transfer the label to the protein fusion;

R² is as above;

R³ is a proton;
and
label is as above.

3. A method according to claim 1 wherein the label is an affinity tag, a first member of a specific binding pair which is capable of specifically binding to the second member of the specific binding pair, a molecule which is attachable to a solid phase, a spectroscopic label, a molecule which is capable of generating reactive radicals, a candidate compound or library of candidate compounds, a molecule which is capable of crosslinking to other biomolecules, a nucleic acid or a derivative thereof capable of undergoing base-pairing with its complementary strand, a lipid, or a hydrophobic molecule with membrane-inserting properties, preferably wherein the label is an affinity tag, a first member of a specific binding pair which is capable of specifically binding to the second member of the specific binding pair, or a molecule which is attachable to a solid phase.

4. A method according to claim 1 wherein it comprises detecting the labelled fusion protein in a cell culture system.

5. A method according to claim 4 wherein it further comprises initially transforming the cells with an expression vector comprising nucleic acid encoding the fusion protein linked to control sequences to direct its expression.

6. A method according to any of claims 1 to 5 wherein it further comprises manipulating the labelled fusion protein using a property introduced by the label to the fusion protein.

7. A method according to any of claims 1 to 6 wherein the labelled substrate is

8. A method according to any of claims 2 to 7 wherein, in the second formula, R¹ is a substituted or unsubstituted alkyl chain, a substituted or unsubstituted cycloalkyl group with a ring size between three and ten carbons, a substituted or unsubstituted heterocycle with a ring size between three and ten carbons, or a substituted or unsubstituted aromatic heterocycle with a ring size between three and ten carbons.

9. A method according to any of claims 2 to 8 wherein the linker group R² is a substituted or unsubstituted alkyl chain or a polyethylene glycol.

10. A method according to any of claims 1 to 9 wherein the label is biotin, avidin or streptavidin.

11. A method according to any of claims 1 to 9 wherein the label is linked to the fusion protein via a cleavable linker so that the fusion protein can be released from the label.

12. A method according to any of claims 1 to 9 wherein the label is linked to the fusion protein via a photocleavable linker so that the fusion protein can be released from the label.

13. A method according to claim 3 wherein it further comprises contacting the fusion protein with a molecule comprising the second member of the specific binding pair.

14. A method according to claim 3 or claim 13 wherein the first and second members of the specific binding pair are a first and second protein, an antibody and antigen, an enzyme and substrate or a ligand and receptor.

15. A method according to claim 3 wherein it further comprises contacting the labelled fusion protein with a solid support so that it becomes immobilised on the solid support.

Drawing